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Journal of Lipid Research, Vol. 50, 2124-2130, October 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS

Akira Honda^*,, Kouwa Yamashita, Tadashi Ikegami^**, Takashi Hara, Teruo Miyazaki^*,, Takeshi Hirayama^**, Mitsuteru Numazawa and Yasushi Matsuzaki^1,,**

^* Center for Collaborative Research, Tokyo Medical University Ibaraki Medical Center, Ami, Ibaraki 300-0395, Japan
Department of Development for Community Medicine, Tokyo Medical University Ibaraki Medical Center, Ami, Ibaraki 300-0395, Japan
^** Department of Gastroenterology, Tokyo Medical University Ibaraki Medical Center, Ami, Ibaraki 300-0395, Japan
Faculty of Pharmaceutical Science, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan
Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan

¹ To whom correspondence should be addressed. e-mail: ymatsuzaki-gi{at}umin.ac.jp

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 µl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 µl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 ± 1.1 pmol, which was in complete agreement with the observed ₀ = 15.0 ± 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

Supplementary key words acetyl-CoA carboxylase • carnitine palmitoyl transferase 1 • fatty acid synthase • liquid chromatography-electrospray ionization-tandem mass spectrometry • malonic acid • malonyl-CoA • malonyl-CoA decarboxylase

Abbreviations: ACC, acetyl-CoA carboxylase; DMP-MA, Di-(1-methyl-3-piperidinyl)malonate; FAS, fatty acid synthase; MA, malonic acid (malonate); MCD, malonyl-CoA decarboxylase; MMA, methylmalonic acid (methylmalonate); N-ESI, ESI in negative mode; P-ESI, ESI in positive mode; SA, succinic acid (succinate); SRM, selected reaction monitoring

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